mesothelin as a biomarker for targeted therapy...expression is generally limited to normal...

REVIEW Open Access

Mesothelin as a biomarker for targetedtherapyJiang Lv1,2,3 and Peng Li1,2*

Abstract

CAR-T cell therapy targeting CD19 has achieved remarkable success in the treatment of B cell malignancies, whilevarious solid malignancies are still refractory for lack of suitable target. In recent years, a large number of studieshave sought to find suitable targets with low “on target, off tumor” concern for the treatment of solid tumors.Mesothelin (MSLN), a tumor-associated antigen broadly overexpressed on various malignant tumor cells, while itsexpression is generally limited to normal mesothelial cells, is an attractive candidate for targeted therapy. Strategiestargeting MSLN, including antibody-based drugs, vaccines and CAR-T therapies, have been assessed in a largenumber of preclinical investigations and clinical trials. In particular, the development of CAR-T therapy has showngreat promise as a treatment for various types of cancers. The safety, efficacy, doses, and pharmacokinetics ofrelevant strategies have been evaluated in many clinical trials. This review is intended to provide a brief overview ofthe characteristics of mesothelin and the development of strategies targeting MSLN for solid tumors. Further, wediscussed the challenges and proposed potential strategies to improve the efficacy of MSLN targetedimmunotherapy.

Keywords: Mesothelin, Biomarker, Targeted therapy, Immunotherapy, CAR-T

BackgroundThe discovery and function of MSLNThe MSLN gene encodes a 71-KD precursor, which is aglycosylphosphatidylinositol (GPI)-anchored membraneglycoprotein that is cleaved into two products at arginine295 (Arg295): a soluble 31-KD N-terminal protein calledmegakaryocyte potentiating factor (MPF) and a 40-KDmembrane-bound fragment called MSLN (mesothelin).Both MPF and MSLN are bioactive, but their exact func-tions remain unclear. MPF was initially reported tostimulate megakaryocyte colony formation in the pres-ence of interleukin-3 in mice but not alone [1], while itsactivity is unknown in humans. MSLN was first de-scribed as a membrane protein expressed on mesotheli-oma and ovarian cancer cells [2] and normal mesothelialcells [2, 3]. A previous study showed that MSLN seemed

to be a nonessential component in normal cells, asMSLN knockout mice did not present with abnormaldevelopment or reproduction [4]. In contrast, preclinicaland clinical studies showed that aberrant MSLN expres-sion on tumor cells plays an important role in promot-ing proliferation and invasion [5]. MSLN has also beenidentified as a receptor of CA125 that mediates celladhesion [6]. The interaction of CA125 and MSLN playan important role in ovarian cancer cell peritonealimplantation and increase the motility and invasion ofpancreatic carcinoma cells [7–9]. The overexpression ofMSLN could activate the NFκB, MAPK, and PI3K path-ways and subsequently induce resistance to apoptosis[10] or promote cell proliferation, migration, and metas-tasis by inducing the activation and expression of MMP7[9] and MMP9 [5]. An increase in tumor burden andpoor overall survival are associated with elevated MSLNexpression according to clinical observations [11, 12].Structural prediction revealed that a superhelical struc-ture with armadillo-type repeats constitutes a part of itsthree-dimensional structure [13], and the structure of anN-terminal fragment that binds to the Fab SS1 antibody

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: [email protected] Laboratory of Regenerative Biology, South China Institute for Stem CellBiology and Regenerative Medicine, Guangzhou Institutes of Biomedicineand Health, Chinese Academy of Sciences, Guangzhou, China2Guangdong Provincial Key Laboratory of Stem Cell and RegenerativeMedicine, South China Institute for Stem Cell Biology and RegenerativeMedicine, Guangzhou Institutes of Biomedicine and Health, ChineseAcademy of Sciences, Guangzhou, ChinaFull list of author information is available at the end of the article

Lv and Li Biomarker Research (2019) 7:18 https://doi.org/10.1186/s40364-019-0169-8

http://crossmark.crossref.org/dialog/?doi=10.1186/s40364-019-0169-8&domain=pdfhttp://orcid.org/0000-0001-6064-3882http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/publicdomain/zero/1.0/mailto:[email protected]

has been clarified [14], but the structure of the wholeprotein is still unclear.

Expression of MSLN in malignant cells and prognosisGenerally, MSLN is expressed on normal mesothelialcells in the pleura, pericardium, and peritoneum and inepithelial cells on the surface of the ovary, tunica vagina-lis, rete testis, and fallopian tubes in trace amounts [3].In contrast, the aberrant overexpression of MSLN isobserved in various cancer cells. MSLN was initiallycharacterized in mesothelioma and ovarian cancer byChang et al. with the mAb K1 [15]. Chang and col-leagues found that MSLN was present in 10 of 15 non-mucinous ovarian cancers and absent in all 4 mucinousovarian cancers examined [2]. In addition, all 15 cases ofepithelial mesothelioma, but none of the 4 cases ofsarcomatous mesothelioma, expressed MSLN [16]. Thiswas in line with the results of another independent studythat confirmed MSLN reactivity in all 44 epithelioidmesotheliomas and in the epithelial components of 3biphasic mesotheliomas, but not in any of 8 sarcomatousmesotheliomas examined [17]. According to the statis-tics in this study, MSLN was present in 15 of 48 (31%)lung cancers (adenocarcinomas (12/31) and squamouscarcinomas (limited, 3/17)) and in 42 of 86 (49%) non-pulmonary adenocarcinomas (ovary (14/14), peritoneum(5/5), endometrium (6/9), pancreas (10/11), stomach (2/4), and colon (5/16); none of 12 breast, 9 kidney, 4 thy-roid, and 2 prostate cancers showed evidence of MSLN)according to assays with the 5B2 anti-MSLN monoclonalantibody. MSLN was immunohistochemically evaluatedin 596 lung carcinomas of different types by MiettinenM and Sarlomo-Rikala M in 2003 [18]. MSLN reactivitywas observed in 78 of 148 (53%) adenocarcinomas, 29 of124 (23%) squamous cell carcinomas and 15 of 118(13%) large cell carcinomas but was absent in small cellcarcinomas. These results suggest that MSLN could actas an immunohistochemical biomarker for the determin-ation of the subtype classification of mesotheliomas andlung cancer to a certain degree because of its specificexpression pattern in these two cancers. MSLN isexpressed in the majority of pancreatic cancers, andindependent studies revealed that almost 100% ofpancreatic cancers are positive for MSLN but thatnormal pancreatic tissues did not show evidence ofMSLN [3, 19, 20]. Subsequent studies demonstratedthe expression of MSLN in a broad spectrum of solidtumors with distinct frequency and distribution patterns,including extrahepatic biliary cancers (95%), triple nega-tive breast cancer (66%), endometrial carcinomas (59%),colorectal carcinomas (30%), cervical carcinomas (25%)and esophageal (46%), endometrial (89%) and thymiccancer [3, 21–26]. A recent study reported that 25.6% of117 patients with gastric carcinoma showed high levels of

MSLN expression, which was associated with a poor prog-nosis [27]. We also detected MSLN expression to differentdegrees in 9 gastric cancer tissues but not in normalgastric tissue [28]. The elevated expression of MSLN wascorrelated with poorer prognoses in patients with ovariancancer [29], cholangiocarcinoma [30, 31], lung adenocar-cinoma [29, 32], triple negative breast cancer [4, 33] andresectable pancreatic adenocarcinoma [34–36].In addition, MSLN is shed into the serum of patients

with solid tumors, in which it is referred to as solubleMSLN-related protein (SMRP) [37]. The production ofSMRP could be associated with abnormal splicing, whichresults in a secreted form or its cleavage from the mem-brane by the TNFα-converting enzyme ADAM17 [38].SMRP was also identified as a promising cancer bio-marker in the sera of patients with mesothelioma, inwhich elevated SMR levels in serum was correlated withadvanced stage and increased disease burden [37, 39].However, the sensitivity and specificity of SMRP as atumor marker in ovarian cancer was limited [40]. Thevalue of soluble MSLN in diagnosis and the predictionof cancer progression remains to be determined, and itscombination with other tumor markers may be moreprecise for diagnosis.

Targeted therapyGiven that MSLN expression is rather limited in severalnormal tissues but highly elevated in the solid tumorsmentioned above, MSLN is a potential target for anti-gen-specific therapy (Fig. 1).

Antibody-based drugsAntibody-based drugs are used to target and kill tumorcells via neutralization by antibodies, antibody-dependentcell-mediated cytotoxicity (ADCC), antibody-dependentcell-mediated phagocytosis (ADCP) or antibodies conju-gated with effector molecules (toxins or inhibitors), whichmediate apoptosis or suppress cell proliferation.The specific uptake of the indium111−labeled MSLN

antibody K1 by tumor cells was observed by Hassen etal. [41]. The conjugation of a fragment of Pseudomonasexotoxin A (PE) to this antibody resulted in cytotoxicityin MSLN-expressing cell lines and tumor regression intumor-bearing mice [42]. A new murine-derived anti-body with higher affinity termed SS1 was produced viaphage display and hotspot mutagenesis [43, 44]. The fu-sion of the PE38 portion to SS1 resulted in a recombin-ant immunotoxin (RIT) termed SS1P, which enters cellsby receptor-mediated endocytosis and induces apoptosisby inactivating elongation factor 2 to impede proteinsynthesis [45]. Many drugs based on the MSLN anti-body SS1 or other modified and humanized versionshave been developed for targeted therapy (Table 1).

Lv and Li Biomarker Research (2019) 7:18 Page 2 of 18

SS1PSS1P has been tested in several clinical trials that en-rolled patients with advanced cancers. In an early phaseI clinical trial (NCT00066651) [48], the dose-limitingtoxicities (DLTs), maximum tolerated dose (MTD) andpharmacokinetics (PK) of SS1P were tested in 34 patientswith mesothelioma (n = 20), ovarian cancer (n = 12) andpancreatic cancer (n = 2). With a limited sample size, thisclinical trial demonstrated that the combination of SS1Pwith prednisone can reduce the risk of toxicity due toSS1P and allow the use of an increased drug dosage. Nosignificant pericardial toxicity was observed in any of thepatients, which suggested that the MSLN antibody SS1Ppresented less risk to pericardial mesothelial cells. Amongthe 33 evaluable patients, 4 had a partial response (PR), 19had stable disease (SD), and 10 had progressive disease(PD). However, SS1P was proven to be immunogenic in alater clinical trial (NCT00006981) [49]. Twenty-fourpatients with chemo-resistant solid tumors received SS1Ptreatment at dosages of 4, 8, 12, 18, and 25 μg/kg/day(× 10). One patient had a PR, 12 had SD, and 11 had

PD. It is noteworthy that high levels of neutralizingantibodies against SS1P were detected in 75% of patients,which could undermine the anti-tumor efficacy.Given that the administration of SS1P alone showed a

moderate effect, the combination therapy might be moreeffective. In the clinical trial NCT01362790 [47], 10patients with chemotherapy-refractory mesotheliomareceived SS1P in combination with pentostatin and cyclo-phosphamide. Three patients had a PR (44% ~ 74%), 3 hadSD and 4 had PD. Adverse events were evaluated for allpatients. Grade 3 toxicities, including noncardiac chestpain, pleuritic pain, and back pain (9% each) were ob-served, but no grade 4 toxicities were observed in patients.Meanwhile, adverse events associated with pentostatin orcyclophosphamide, such as grade 4 lymphopenias, wereobserved in all patients. In contrast to the trials describedabove, the involvement of pentostatin and cyclophospha-mide delayed the formation of neutralizing antibodies toSS1P, thereby allowing a prolonged period of therapy.SS1P combined with pemetrexed and cisplatin was furthertested for treating chemotherapy-naive patients with

Fig. 1 MSLN-targeted therapy strategies. a, the precursor protein is cleaved into two products, i.e. soluble protein MPF and GPI-anchoredmembrane protein MSLN; b, anti-MSLN antibody derived scFv, Fab, or intact/modified antibody are conjugated with the effector molecules(inhibitor or toxin) and induce cell death after binding to tumor cells; c, the binding of amatuximab to MSLN expressed on tumor cell membraneleads to ADCC; d, HPN536 directs T cells to kill tumor cells expressing MSLN; e, cancer vaccines arouse tumor specific immune response; f, the Tcells are engineering to express CAR and redirected to tumor cells


advanced malignant pleural mesothelioma (MPM)(NCT01445392) [46]. Of the 20 evaluable subjects, 12 pa-tients had a PR, 3 had SD, and 5 had PD. Notably, thechanges in the relative serum levels of MSLN, MPF andCA125 were significantly correlated with responses (PR >SD > PD). These biomarker levels were generally de-creased in 12 patients who received PR but were substan-tially increased in 5 patients who had PD.

AmatuximabAmatuximab (MORAb-009) is a chimeric monoclonalantibody consisting of the SS1 scFv fused to the humanIgG1 and κ constant regions. The binding of amatuxi-mab to MSLN expressed on tumor cell surfaces leads toADCC.Two clinical trials (NCT00570713 and NCT00738582)

showed that no severe (grade 3 or 4) drug hypersensitiv-ity adverse events (DHAEs) were observed in any of thesubjects. Among the 20 of 24 patients evaluable for re-sponse, none had complete or partial responses, andonly 11 patients had SD and 9 had PD [52]. MORAb-009 treatment resulted in a remarkable elevation inserum CA125 levels in all 8 patients under surveillance,possibly due to the interruption of binding betweenMSLN and CA125 by amatuximab, which could pre-vent the intraperitoneal/intrapleural metastasis ofovarian cancer and mesothelioma [53]. A clinical trial(NCT01018784) in Japanese patients with mesotheli-oma, pancreatic adenocarcinoma or other MSLN-posi-tive solid tumors revealed that the weekly singleadministration of amatuximab in 4-week cycles at in-creasing doses ranging from 50 to 200 mg/m2 led tolimited treatment effects. Three of the 17 patientshad SD, and 14 had PD [51].The anti-tumor effect of amatuximab in combination

with pemetrexed and cisplatin was elevated in 89 pa-tients at 26 centers (NCT00738582) [54]. Amatuximabin combination with pemetrexed and cisplatin was ad-ministered according to the response (PR or SD) for upto 6 cycles. Thirty-three patients had a PR, and 42 hadSD. The detection of the change in the MPF level inserum before and after treatment in 59 patients alsoshowed that the decreased MPF level was correlatedwith good prognosis. The combination therapy led to se-vere adverse events, including hypersensitivity reactions,neutropenia, and atrial fibrillation. Dyspnea and fatiguewere observed during the maintenance phase.An 111Indium (111In) radiolabel was used to characterize

the biodistribution and dosimetry of amatuximab in 6patients (4 with malignant mesothelioma and 2 withpancreatic adenocarcinoma) [60]. SPECT/CT imagingshowed 111In-amatuximab uptake in both primarytumors and metastatic sites and that uptake was in-creased in mesothelioma compared with that in

pancreatic cancer. Notably, 111In-amatuximab uptake inthe heart, liver, kidneys and spleen was also confirmed.Even so, amatuximab was generally well tolerated.Amatuximab PK was characterized in the clinical trialNCT02357147. It revealed that higher amatuximab expos-ure in combination with chemotherapy was associatedwith prolonged OS [50].

Anetumab ravtansineAnetumab ravtansine, also referred to as BAY94–9343,is a human anti-MSLN antibody fused to DM4, which isa maytansinoid tubulin inhibitor that mainly affectsproliferating cells. The specific binding of BAY94–9343to MSLN with high affinity induces efficient antigeninternalization. BAY94–9343 showed dose-dependentanti-tumor efficacy and bystander effects in xenogeneictumor models [56]. The antitumor efficacy of anetumabravtansine in combination with pegylated liposomaldoxorubicin (PLD), carboplatin, copanlisib and bevacizu-mab was investigated for the treatment of ovarian can-cer. The involvement of combination therapy showedenhanced anti-proliferative activity and increased apop-tosis in vitro and improved in vivo efficacy in tumor-bearing mice [55]. The safety, tolerability, pharmacokin-etics, and pharmacodynamics were then evaluated inclinical trials. Several phase 1/2 studies were carried outto explore the dosage and side effects of anetumab rav-tansine when administered together with pemetrexed,cisplatin, PLD, itraconazole, gemcitabine, pembrolizu-mab, atezolizumab, gemcitabine hydrochloride, ipilimu-mab or nivolumab (Table 1). However, only one clinicaltrial data for anetumab ravtansine was submitted toClinicalTrials.gov prior to the submission of this review.

DMOT4039ADMOT4039A is a humanized anti-MSLN mAb(h7D9.v3) fused to the antimitotic agent monomethylauristatin E (MMAE) [61]. It inhibited cell proliferationat an IC50 of 0.3 nmol/L and regressed tumor growth ina dose-dependent manner in a mouse model. In anotherclinical trial (NCT01469793), DMOT4039A was admin-istered to 71 patients with pancreatic cancer (n = 40) orovarian cancer (n = 31) [62]. Fifty-four patients receiveda DMOT4039A injection every 3 weeks (2.4–2.8 mg/kg;q3w), and 17 patients received an injection weekly (0.8–1.2 mg/ kg). Hyperglycemia (grade 3) and hypophospha-temia (grade 3) were observed in 2 patients treated withDMOT4039A every 3 weeks at a dosage of 2.8 mg/kgbut no DLTs were observed in patients treated withother dosages. Related severe adverse events occurred in5 patients at a dosage of 2.4–2.8 mg/kg every 3 weeksand one patient at a dosage of 1.2 mg/kg weekly. Cumu-lative peripheral neuropathy (grades 1–3) was observedin 14 patients due to microtubule inhibitors. Six patients


https://clinicaltrials.gov/

Table

1ClinicaltrialsforMSLN-targe

tedtherapiesbasedon

antib

ody-baseddrug

sandvaccines

Age

ntNCTNum

ber

(Referen

ce)

Title

Status

(Results)

Interven

tions

Phases

Enrollm

ent

Start

Date

Locatio

ns

SS1P

NCT01445392

[46]

SS1(dsFV)PE38Plus

Pemetrexedand

Cisplatin

toTreatMalignant

Pleural

Mesothe

lioma

Term

inated

Biolog

ical:M

ulticycleSS1P

Drug:

Pemetrexed

Drug:

Cisplatin

Biolog

ical:SinglecycleSS1PBiolog

ical:

MulticycleSS1P

Drug:

Pemetrexed

Drug:

Cisplatin

Biolog

ical:SinglecycleSS1PBiolog

ical:

MulticycleSS1P

Drug:

Pemetrexed

Drug:

Cisplatin

Biolog

ical:SinglecycleSS1P

Phase

124

2007-

11-14

UnitedStates

NCT01362790

[47]

SS1P

andPentostatin

Plus

Cycloph

osph

amide

forMesothe

lioma

Unkno

wn

status

(Results

Subm

itted

)

Drug:

Pentostatin

;Drug:

Cycloph

osph

amide;

Biolog

ical:SS1(dsFv)PE38

-lot073I0809;

Biolog

ical:SS1(dsFv)PE38

-lotFIL129J01

Phase

1/2

552011-

05-11

UnitedStates

NCT01051934

APh

aseITrialo

fSS1(dsFv)

PE38

With

Paclitaxel,Carbo

platin,and

Bevacizumab

inSubjectsWith

UnresectableNon

-SmallC

ell

Lung

Ade

nocarcinom

a

Com

pleted

Drug:

SS1(dsFv)

PE38;D

rug:

Paclitaxel;

Drug:

Carbo

platin;D

rug:

Bevacizumab

Phase

12

2009-

12-29

UnitedStates

NCT00066651

[48]

Immun

otoxin

Therapyin

Treatin

gPatients

With

AdvancedSolid

Tumors

Com

pleted

Biolog

ical:SS1(dsFv)-PE38im

mun

otoxin

Phase

12003-

07-01

UnitedStates

NCT00006981

[49]

Immun

otoxin

Therapyin

Treatin

gPatients

With

AdvancedCancer

Com

pleted

Biolog

ical:SS1(dsFv)-PE38im

mun

otoxin

Phase

12000-

12-01

UnitedStates

Amatuxim

abNCT02357147

[50]

Stud

yof

theSafety

andEfficacyof

Amatuxim

abin

Com

binatio

nWith

PemetrexedandCisplatin

inSubjectsWith

UnresectableMalignant

Pleural

Mesothe

lioma(M

PM)

Term

inated

Drug:

Placeb

o;Drug:

Amatuxim

ab;

Drug:

Pemetrexed;

Drug:

Cisplatin

Phase

2108

2015-

11-03

Australia;France;

Germany;Italy;

UnitedKing

dom;

UnitedStates

NCT01521325

ASing

le-DosePilotStud

yof

Radiolabeled

Amatuxim

ab(M

ORA

b-009)

inMesothe

linOver

Expressing

Cancers

Com

pleted

Drug:

Amatuxim

abPh

ase

16

2011-

09-01

UnitedStates

NCT01413451

Amatuxim

abforHighMesothe

linCancers

Term

inated

Drug:

Amatuxim

ab(M

ORab-009)

Early

Phase

1

72011-

07-12

UnitedStates

NCT01018784

[51]

AStud

yof

MORA

b-009in

PatientsWith

Solid

Tumor

Com

pleted

Drug:

MORA

b-009

Phase

117

2009-

11-01

Japan

NCT00738582

[52–54]

AnEfficacyStud

yof

MORA

b-009(Amatuxim

ab)

inSubjectsWith

PleuralM

esothe

lioma

Com

pleted

(Results

Subm

itted

)

Drug:

MORA

b-009(Amatuxim

ab);Drug:

Pemetrexed;

Drug:

Cisplatin

Phase

289

2008-

12-01

Canada;Germany;

Nethe

rland

s;Spain;

UnitedStates

NCT00570713

[52]

AnEfficacyStud

yof

MORA

b-009in

Subjects

With

PancreaticCancer

Com

pleted

(Results

Available)

Drug:

MORA

b-009;Drug:

Placeb

o;Drug:

Placeb

o;Drug:

Gem

citabine

Phase

2155

2007-

12-01

Belgium;C

anada;

Germany;Spain;

UnitedStates


Table


tedtherapiesbasedon

antib

ody-baseddrug

sandvaccines

(Con

tinued)

Age

ntNCTNum

ber

(Referen

ce)

Title

Status

(Results)

Interven

tions

Phases

Enrollm

ent

Start

Date

Locatio

ns

NCT00325494

AStud

yof

MORA

b-009in

SubjectsWith

PancreaticCancer,Mesothe

lioma,or

Certain

Type

sof

Ovarianor

Lung

Cancer

Com

pleted

Drug:

MORA

b-009

Phase

124

2006-

05-01

UnitedStates

Ane

tumab

ravtansine

(BAY94–9343)

NCT03816358

Ane

tumab

Ravtansine

With

Nivolum

ab,

Ipilimum

abandGem

citabine

Hydrochlorid

ein

Treatin

gPatientsWith

Mesothe

linPo

sitive

AdvancedPancreaticCancer

Suspen

ded

Biolog

ical:A

netumab

Ravtansine

;Drug:

Gem

citabine

Hydrochlorid

e;Biolog

ical:

Ipilimum

ab;Biological:Nivolum

ab

Phase

164

2019-

07-01

Canada

NCT03455556

Ane

tumab

Ravtansine

andAtezolizum

abin

Treatin

gParticipantsWith

Advanced

Non

-smallC

ellLun

gCancer

Recruitin

gBiolog

ical:A

netumab

Ravtansine

;Biological:

Atezolizum

ab;O

ther:LaboratoryBiom

arker

Analysis

Phase

1/2

492018-

08-10

UnitedStates

NCT03126630

Pembrolizum

abWith

orWith

outAne

tumab

Ravtansine

inTreatin

gPatientsWith

Mesothe

lin-Positive

PleuralM

esothe

lioma

Recruitin

gBiolog

ical:A

netumab

Ravtansine

;Other:

Labo

ratory

Biom

arkerAnalysis;Biolog

ical:

Pembrolizum

ab;O

ther:Pharm

acolog

icalStud

y

Phase

1/2

134

2018-

02-08

UnitedStates;C

anada

NCT03102320

Phase1b

Multi-indicatio

nStud

yof

Ane

tumab

Ravtansine

inMesothe

linExpressing

Advanced

Solid

Tumors

Recruitin

gDrug:

Cisplatin;D

rug:

Gem

citabine

;Drug:

Ane

tumab

ravtansine

(BAY94–9343)

Phase

1348

2017-

05-26

UnitedStates;

Australia;Belgium

;Canada;France;

Germany;Italy;

Korea;Nethe

rland

s;Sing

apore;Spain;

Switzerland

;UnitedKing

dom

NCT03023722

PhaseIIAne

tumab

Ravtansine

inPre-treated

Mesothe

lin-expressingPancreaticCancer

Recruitin

gDrug:

anetum

abravtansine

Phase

230

2017-

05-11

UnitedStates

NCT02824042

Thorou

ghEC

G(Electrocardiogram

)and

DrugInteractionStud

yWith

Ane

tumab

Ravtansine

andItracon

azole

Active,no

trecruitin

gDrug:

Ane

tumab

ravtansine

(BAY94–9343);

Drug:

Itracon

azole

Phase

163

2016-

09-12

UnitedStates;

Australia;Belgium

;France;N

ethe

rland

s;Spain

NCT02839681

Anti-M

esothe

linAntibod

yDrugCon

jugate

Ane

tumab

Ravtansine

forMesothe

linExpressing

Lung

Ade

nocarcinom

a

Term

inated

(Results

Subm

itted

)

Drug:

Ane

tumab

Ravtansine

;Device:

Bloo

dtest

Phase

22

2016-

07-19

UnitedStates

NCT02751918

[55]

PhaseIb

Stud

yof

Ane

tumab

Ravtansine

inCom

binatio

nWith

PegylatedLipo

somal

Doxorub

icin

inPatientsWith

Recurren

tMesothe

lin-expressingPlatinum

-resistant

Cancer

Recruitin

gDrug:

Ane

tumab

ravtansine

(BAY94–9343);

Drug:

PegylatedLipo

somalDoxorub

icin

Phase

171

2016-

06-08

UnitedStates;

Belgium;France;Japan;

Moldo

va;Spain

NCT02696642

PhaseIStudy

ofAne

tumab

Ravtansine

inHep

aticor

RenalImpairm

ent

Active,no

trecruitin

gDrug:

Ane

tumab

ravtansine

(BAY94–9343)

Phase

154

2016-

04-14

France;M

oldo

va

NCT02639091

PhaseIb

Stud

yof

Ane

tumab

Ravtansine

inCom

binatio

nWith

Pemetrexedand

Cisplatin

inMesothe

lin-expressingSolid

Tumors

Active,no

trecruitin

gDrug:

BAY94–9343;Drug:

Pemetrexed;

Drug:

Cisplatin

Phase

136

2016-

02-03

UnitedStates;Italy


Table


tedtherapiesbasedon

antib

ody-baseddrug

sandvaccines

(Con

tinued)

Age

ntNCTNum

ber

(Referen

ce)

Title

Status

(Results)

Interven

tions

Phases

Enrollm

ent

Start

Date

Locatio

ns

NCT02610140

PhaseIIAne

tumab

Ravtansine

as2n

dLine

Treatm

entforMalignant

Pleural

Mesothe

lioma(M

PM)

Active,no

trecruitin

gDrug:

Ane

tumab

ravtansine

(BAY94–9343);

Drug:

Vino

relbine

Phase

2248

2015-

12-03

UnitedStates;

Australia;Belgium

;Canada;Finland;

France;Italy;Korea;

Nethe

rland

s;Po

land

;Ru

ssianFede

ratio

n;Spain;Turkey;

UnitedKing

dom

NCT02485119

PhaseID

oseEscalatio

nStud

yof

BAY94–9343

Given

byIntraven

ousInfusion

Every3Weeks

inJapane

seSubjectsWith

Advanced

Malignancies

Com

pleted

Drug:

BAY94–9343

Phase

112

2015-

08-14

Japan

NCT01439152

[56]

PhaseIStudy

toDeterminetheMaxim

umTolerableDoseof

BAY94–9343

inPatients

With

AdvancedSolid

Tumors.

Active,no

trecruitin

gDrug:

BAY94–9343;D

rug:

BAY94–9343

(Expansion

);Drug:

BAY94–9343

(1.8mg/kg);

Drug:

BAY94–9343

(2.2mg/kg)

Phase

1147

2011-

09-07

UnitedStates

DMOT4039A

NCT01469793

AStud

yof

DMOT4039A

inParticipants

With

UnresectablePancreaticor

Platinum

-Resistant

OvarianCancer

Com

pleted

Drug:

DMOT4039A

Phase

171

2011-

11-01

UnitedStates

NCT01832116

89Zr-M

MOTPETIm

agingin

Pancreatic

andOvarianCancerPatients

Com

pleted

Drug:

89Zr-M

MOT0530A

Phase

111

2013-

03-01

Nethe

rland

s

BMS-986148

NCT02884726

[57]

Phase1Stud

yof

Mesothe

lin-ADC

Com

pleted

Drug:

BMS-986148

Phase

18

2016-

10-14

Japan

NCT02341625

[57]

AStud

yof

BMS-986148

inPatientsWith

Select

AdvancedSolid

Tumors

Active,no

trecruitin

gDrug:

BMS-986148;Biological:Nivolum

abPh

ase

1/2

407

2015-

06-17

UnitedStates;

Australia;Belgium

;Canada;Italy;

Nethe

rland

s;UnitedKing

dom

LMB-100

NCT03644550

Anti-M

esothe

linIm

mun

otoxin

LMB-100

Followed

byPembrolizum

abin

Malignant

Mesothe

lioma

Recruitin

gDrug:

LMB-100;Biolog

ical:Pem

brolizum

abPh

ase

238

2018-

12-04

UnitedStates

NCT03436732

Mesothe

lin-Targe

tedIm

mun

otoxin

LMB-

100in

Com

binatio

nWith

SEL-110in

SubjectsWith

Malignant

Pleuralo

rPeriton

ealM

esothe

lioma

Suspen

ded

Drug:

LMB-100;Drug:

SEL-110

Phase

123

2018-

02-28

UnitedStates

NCT02810418

Mesothe

lin-Targe

tedIm

mun

otoxin

LMB-100

Alone

orin

Com

binatio

nWith

Nab-Paclitaxel

inPeop

leWith

PreviouslyTreatedMetastatic

and/or

Locally

AdvancedPancreaticDuctal

Ade

nocarcinom

aandMesothe

linExpressing

Solid

Tumors

Active,no

trecruitin

gDrug:

LMB-100;Drug:

Nab-Paclitaxel;

Device:Mesothe

linExpression

Phase

1/2

402016-

08-03

UnitedStates

NCT02798536

Mesothe

lin-Targe

tedIm

mun

otoxin

LMB-100in

Peop

leWith

Malignant

Mesothe

lioma

Active,no

trecruitin

gDrug:

LMB-100;Drug:

nab-paclitaxel

Phase

121

2016-

06-10

UnitedStates


Table


tedtherapiesbasedon

antib

ody-baseddrug

sandvaccines

(Con

tinued)

Age

ntNCTNum

ber

(Referen

ce)

Title

Status

(Results)

Interven

tions

Phases

Enrollm

ent

Start

Date

Locatio

ns

BAY2287411

NCT03507452

First-in-hum

anStud

yof

BAY2287411

Injection,

aThorium-227

Labe

ledAntibod

y-chelator

Con

jugate,inPatientsWith

TumorsKn

ownto

ExpressMesothe

lin

Recruitin

gDrug:

BAY2287411

Phase

1228

2018-

06-13

UnitedStates;Finland

;Nethe

rland

s;Sw

eden

;UnitedKing

dom

HPN

536

NCT03872206

Stud

yof

HPN

536in

PatientsWith

Advanced

CancersAssociatedWith

Mesothe

linExpression

Recruitin

gBiolog

ical:H

PN536

Phase

1/2

872019-

04-16

UnitedStates

CRS-207

NCT02004262

Safety

andEfficacyof

Com

binatio

nListeria/GVA

XPancreas

Vaccinein

the

PancreaticCancerSetting

Com

pleted

(Results

Available)

Biolog

ical:G

VAXPancreas

Vaccine;Biolog

ical:

CRS-207;D

rug:

Che

mothe

rapy;D

rug:

cyclop

hosphamide

Phase

2303

2014-

02-05

Canada

NCT01417000

[58]

Safety

andEfficacyof

Com

binatio

nListeria/GVA

XIm

mun

othe

rapy

inPancreaticCancer

Com

pleted

(Results

Available)

Biolog

ical:G

VAXPancreas;Biological:CRS-207;

Drug:

Cycloph

osph

amide

Phase

293

2011-

09-21

UnitedStates

NCT00585845

[59]

Stud

yof

Safety

andTolerabilityof

Intraven

ous

CRS-207

inAdu

ltsWith

Selected

Advanced

Solid

TumorsWho

HaveFailedor

Who

Are

Not

Candidates

forStandard

Treatm

ent

Term

inated

Biolog

ical:C

RS-207,Live-attenu

ated

Listeria

mon

ocytogenes

expressing

human

Mesothe

linPh

ase

117

2007-

12-01

UnitedStates;Israel

JNJ-64041757

NCT03371381

AnEfficacyandSafety

Stud

yof

JNJ-64041757,

aLive

Atten

uatedListeriaMon

ocytog

enes

Immun

othe

rapy,inCom

binatio

nWith

Nivolum

abVersus

Nivolum

abMon

othe

rapy

inParticipantsWith

AdvancedAde

nocarcinom

aof

theLung

Term

inated

Biolog

ical:JNJ-64041757;D

rug:

Nivolum

abPh

ase

1/2

122018-

01-02

UnitedStates;

Belgium;Spain

NCT02592967

Safety

&Im

mun

ogen

icity

ofJNJ-64041757,

Live-atten

uatedDou

ble-de

letedListeria

Immun

othe

rapy,inSubjectsWith

Non

Small

CellLun

gCancer

Term

inated

Biolog

ical:JNJ-64041757(Coh

ort1A

and1B);

Biolog

ical:JNJ-64041757(Coh

ort2A

and2B)

Phase

118

2015-

12-02

UnitedStates

Neo

antig

enDNAVaccine

NCT03122106

Neo

antig

enDNAVaccinein

PancreaticCancer

PatientsFollowingSurgicalResectionand

AdjuvantChe

mothe

rapy

Recruitin

gBiolog

ical:Personalized

neoantigen

DNAvaccine;

Device:TD

S-IM

Electrod

eArray

System

;Proced

ure:Perip

heralb

lood

draw

s

Phase

115

2018-

01-05

UnitedStates


(4 ovarian cancer; 2 pancreatic cancer) treated withDMOT4039A at 2.4 to 2.8 mg/kg had a PR.

BMS-986148BMS-986148 is an antibody-drug conjugate that mightbe related to MDX-1204, which contains a MAb conju-gated to the potent alkylating agent duocarmycin(MED2460) and causes cell death after internalization bytarget cells [57]. A clinical trial was carried out to evalu-ate the safety, tolerability, pharmacokinetics, immuno-genicity, antitumor activity and pharmacodynamics ofBMS-986148 administered alone and in combinationwith nivolumab in selected patients with mesotheli-oma, nonsmall cell lung cancer (NSCLC), ovariancancer, pancreatic cancer and gastric cancer. Thisstudy aimed to enroll over 400 patients from 12countries (NCT02341625). Another phase 1 clinicaltrial (NCT02884726) in Japan has been completed.

LMB-100/ RG7787LMB-100/ RG7787 is a re-engineered version of a hu-manized anti-MSLN Fab based on SS1 that is fused to atruncated and deimmunized PE24 moiety with higheractivity and less immunogenicity [63]. LMB-100 inhibitsprotein synthesis [64] and is regulated by the tyrosinekinase DDR1 [65]. The addition of a DDR1 inhibitor re-sulted in the increased shrinkage of tumor xenografts.The antitumor efficacy of LMB-100 for pancreatic can-cer, triple negative breast cancer (TNBC), and gastriccancer has been proven in preclinical studies [63, 66]. Itscombination with actinomycin D [67], Nab-Paclitaxel[68], taxanes [69], and panobinostat [70] enhances itsantitumor activity. LMB-100 is currently undergoingclinical testing in combination therapy in patients withMSLN-positive malignancies.

BAY2287411BAY2287411, a thorium-227-labeled antibody-chelatorconjugate, was administered to patients with tumorsknown to express MSLN to evaluate the safety, tolerabil-ity, maximum tolerated dose, PK, anti-tumor activityand recommended dose for further clinical development(NCT03507452). This phase 1 study started in June2018. More than 200 participants may eventually be en-rolled with nonrandomized allocation. A recent studydemonstrated that the combination of BAY2287411 withthe damage response inhibitors ATRi and PARPi re-sulted in synergistic activity and increased anti-tumor ef-ficacy [71].

HPN536HPN536 is the most recent MSLN-targeting antibody-based drug that is currently in clinical trials. It is aMSLN-targeting TriTAC and includes three domains:

1. an anti-MSLN domain that binds to MSLN-positivecells; 2. an anti-albumin domain antibody that ex-tends its half-life; 3. an anti-CD3ε scFv that engagesT cells [72]. HPN536 activates T cells in the presenceof MSLN and directs T cells to kill cells expressingMSLN. It has a half-life of approximately 5 days andis well tolerated in cynomolgus monkeys subjected toa single treatment at a 10 mg/kg dosage. NCI-H292tumor growth was impeded in mice implanted withhuman PBMCs and treated with HPN536. The associ-ated phase 1/2a trial (NCT03872206) is a multicenter,open-label study designed to evaluate the safety, toler-ability, PK and activity of HPN536 in up to 80 pa-tients with advanced cancers associated with MSLNexpression.The short half-life and the immunogenicity of mur-

ine-derived antibodies and bacterial toxins have lim-ited the efficacy of antibody-based drugs. To addressthese issues, novel humanized or fully human anti-MSLN antibodies and toxins with reduced immuno-genicity need to be developed. Many studies haveattempted to do this. The insertion of a disulfidebond to protect the furin cleavage site of SS1-PE24improves its serum half-life and decreases its toxicity[73]. A study suggested that the involvement of albu-min-binding domains could prolong the half-life andincrease antitumor activity [74]. In addition, the re-moval of B- and T-cell epitopes from RIT led togreatly reduced antigenicity [75, 76]. Fully humanantibodies were also developed and verified in pre-clinical studies [77, 78].

VaccinesCancer vaccines are designed to induce tumor-specificimmune responses in the host. A large number of stud-ies have tested multiple platforms, including peptides,proteins, antigen presenting cells, tumor cells, and viralvectors [79]. The bacterium-based vaccine CRS-207,which uses a live-attenuated Listeria monocytogenes(Lm) strain ANZ-100 (Lm ΔactA/ΔinlB) engineered toexpress human MSLN, has been used to treat MSLN-positive cancers in clinical trials [59]. CRS-207 was eval-uated in 17 subjects (7 with pancreatic ductal carcinoma(PDA), 5 with mesothelioma, 3 with NSCLC, and 2 withovarian cancer) in a dose-escalation study with up to 4doses (NCT00585845). CRS-207 was well tolerated atthe top dose (1 × 109 cfu). Immune activation was con-firmed by a multiplexed serum cytokine assay andphenotype analysis. Thirty-seven percent of subjects sur-vived ≥15 months, but none of them had a PR. CRS-207has also been used in combination with low-dose cyclo-phosphamide and another vaccine, GVAX pancreas,which is derived from an irradiated allogeneic GM-CSFsecreting cell line, in patients with metastatic PDA


(NCT01417000) [58]. Sixty-one patients who receivedCRS-207 and Cy/GVAX had longer overall survival(6.1 months) than 29 patients treated with Cy/GVAXalone (3.9 months). A follow-up study to test the im-mune responses and efficacy produced by the com-bination of CRS-207 and the GVAX pancreas vaccine(with cyclophosphamide) compared to those producedby chemotherapy or CRS-207 alone in adults withpreviously treated metastatic pancreatic adenocarcin-oma was conducted. The overall survival was 3.8months for the cohort treated with Cy/GVAX + CRS-207, 5.4 months for the cohort treated with CRS-207alone, and 4.6 months for the cohort treated withchemotherapy (NCT02004262).JNJ-64041757 (previously referred to as ADU-214)

is a live-attenuated, double-deleted (LADD) Listeriamonocytogenes strain used as a potential treatmentfor NSCLC that was engineered by Aduro Biotech,Inc. in 2014. However, two clinical trials thatattempted to evaluate its efficacy alone or in com-bination with nivolumab were both terminated dueto a lack of clinical benefit (NCT02592967 andNCT03371381). A neoantigen DNA vaccine strategyis currently being evaluated in pancreatic cancer pa-tients following surgical resection and adjuvantchemotherapy in an ongoing phase 1 clinical trial(NCT03122106). Neoantigen DNA vaccines incorpor-ate prioritized neoantigens, and personalized MSLNepitopes will be administered intramuscularly usingthe TDS-IM system. The estimated completion dateof this study is March 2022.Despite the fact that there are few clinical trials of

MSLN-targeted vaccines and the results of these tri-als have been disappointing, many preclinical studiesare still ongoing. One study showed that a cell-basedvaccine, Meso-VAX, in combination with the adeno-associated virus (AAV)-IL-12 increased the numberof MSLN-specific T cells and the levels of anti-MSLN Abs and enhanced tumor clearance activity inmice [80]. The anti-tumor effects of the chimericDNA vaccine CTGF/MSLN (containing an antigen-specific connective tissue growth factor linked towith MSLN) in combination with an anti-CD40 Aband the TLR 3 ligand poly(I:C), which are essentialadjuvants for DC maturation, the immuno-modulatorEGCG and Meso-VAX in combination with (AAV)-IL-12 were proven [81]. Recently, a MSLN-derivedepitope peptide restricted to HLA-A*2402 was shownto be effective in inducing peptide-specific CTLs.The MSLN-10-5 peptide-specific CTL clones showedspecific cytotoxic activity against HLA-A*2402-posi-tive MSLN-expressing pancreatic cancer cells, indi-cating that the peptide-based vaccine is a promisingcandidate for therapy [82].

CAR-T therapyThe development of MSLN-targeting CAR-T cellsChimeric antigen receptor T (CAR-T) cells are designedto target cell surface antigens without MHC restriction.Therefore, the CAR-T cells could be broadly applicablein HLA-diverse allogeneic recipients. The CARs are re-combinant receptors commonly consisting of an extra-cellular antigen recognition domain, which is generallyderived from the single chain variable fragment (scFv) ofantibodies, transmembrane domains that function as an-chors in the cytoplasmic membrane, and an intracellulardomain that transmits T cell activation signals. Thefirst-generation CARs consisted of only one intracellularsignaling domain, which was usually a CD3z chain, andthis was sufficient to initiate T cell activation but pro-duced only short-term proliferative activity and a lowlevel of cytotoxicity. The second-generation CARs hadgreatly improved potency through the incorporation ofanother costimulatory molecule (CD28, 4-1BB, or OX40)[83–85]. Furthermore, our team and other groups demon-strated that the third-generation MSLN-targeting CARscontaining two costimulatory domains (CD28, 4-1BB,TLR2, or DAP10) and a hinge domain were superior interms of cell proliferation, cytotoxicity, persistence andtumor suppression efficacy [86–89]. The latest iteration,the fourth-generation CARs, can additionally secrete cyto-kines or other effector molecules, such as IL-12, IL-15, IL-7, CCL19, or αPD-1, to regulate the immune microenvir-onment [90–95].Because MSLN is a highly specific antigen in several

cancers, CAR-T therapy has been proven to be a prom-ising strategy for the treatment of these cancers. TNBCis intractable due to the lack of an effective targetedtherapy. The presence of MSLN in 67% of TNBCsprovides a candidate target for CAR-T therapy of TNBC[23]. MSLN-directed CAR-T cells were demonstrated toinduce cytotoxicity in MSLN-expressing pancreatic can-cer cells in vivo depending on the MSLN expressionlevel to delay tumor growth and eliminate lung metasta-ses in vivo [96, 97]. Our team previously demonstratedthat MSLN was also a promising target for treating lungcancer and gastric cancer [28, 87]. We proved that third-generation CAR-T could effectively delay tumor growthor even completely eradicate subcutaneous tumors,eliminate pulmonary and intraperitoneal metastases ofgastric cancer cells in mice and prolong survival. Simi-larly, the effectiveness of this targeted strategy has alsobeen proven in bile duct carcinoma [98] and ovariancancer [99].CAR-T cells are generally produced via lentivirus

transduction. The CAR genes are cloned into lentiviralvectors and subsequently integrated into the host T cellgenome, allowing for the stable and permanent expres-sion of the CAR. This method has been widely adopted


because it is simple and reliable. Another method usedfor the stable integration of the CAR gene into the T cellgenome is the piggyBac transposon system. The piggy-Bac transposon system is an efficient nonviral methodfor the genomic engineering of mammalian cells, includ-ing pluripotent stem cells and human T lymphocytes,and its advantages include a large cargo capacity,nonrandom integration and the elimination of virus-associated issues [100]. MSLN-targeting CAR-T cellsengineered by the piggyBac transposon system have beenproven to be cytotoxic to pancreatic cancer cells [97]and bile duct carcinoma cells [98]. To avoid the risks as-sociated with genomic integration, several studies haveproposed that CAR-T cells targeting MSLN could begenerated by RNA electroporation [99, 101]. The expres-sion of the CAR was shown to be detectable 7 days afterelectroporation. Multiple injections of RNA-electropo-rated CAR T cells reduced tumor volumes in mice.However, the CAR is transiently expressed and will becompletely eliminated over time as a result of the deg-radation of the CAR mRNA [99, 101].CAR-T cells are generally administered by systemic

delivery, such as intravenous injection. However, system-ically delivered T cells need to pass through the barrierscreated by multiple tissues before infiltrating into tu-mors. Therefore, inefficient T cell infiltration and shortpersistence are common obstacles for solid tumor ther-apy by CAR-T. A recent preclinical study revealed thatregional intrapleural administration of CAR T cells re-sulted in more robust proliferation and increased antitu-mor efficacy with a long persistence of 200 days in anorthotopic MPM model compared with that induced bysystemically infused T cells [102]. Similarly, we foundthat the regional peritumoral delivery of CAR-T cellsproduced enhanced tumor clearance in a subcutaneousGC model [28]. The subcutaneous tumors in some micein the peritumoral delivery group were completely elimi-nated, whereas a moderate effect was observed in thegroup treated with intravenously injected CAR-T cells.In addition, we found improved T cell infiltration in tu-mors in the peritumoral delivery group. Overall, regionaldelivery might enhance the therapeutic effects, but thisrequires verification in clinical trials. To enhance T cellinfiltration, the MSLN-targeting CAR-T cells were alsoengineered to express CCR2b, a chemokine receptor thatis minimally expressed on T cells, while the CCR2b lig-and CCL2 is highly secreted by MPM [103]. The overex-pression of CCR2b enhanced CAR-T cell cytotoxicity intumor cells and chemotaxis in response to CCL2 invitro. A 12.5-fold increase in T cell infiltration into tu-mors and significantly enhanced tumor clearance wereobserved in mice [103].The tumor immune microenvironment is crucial in

regulating T cell immunosurveillance. The upregulation

of PD-L1 in tumor cells and the expression of inhibitoryreceptors, including PD1, CTLA-4, TIM3, LAG3, and2B4, on T cells always reduces the infiltration of T cellsinto tumors and induces T cell exhaustion [95]. Recentpreclinical studies showed that PD-1/PD-L1 blockade orCRISPR/Cas9-mediated PD-1 disruption could rescueMSLN-targeted CAR-T cell responses in vivo [104, 105].Based on this, CAR-T cells engineered to expressimmune checkpoint antibodies (CTLA-4 and PD-1) orto knock out PD-1 are being evaluated in clinical trials[95] (NCT03030001, NCT03182803, NCT03615313,NCT03545815, and NCT03747965). In addition to beingrestricted by immune checkpoint molecules, the func-tion of T cells is regulated by a variety of cytokines. Thedepletion of IL-10 with a blocking antibody or via theelevation of TNF-α and IL-2 levels by an oncolyticadenovirus enhanced and prolonged the functioning ofMSLN-redirected CAR-T cells [106, 107].MSLN-redirected CAR-T cells are also associated with

the “on target, off tumor” issue. Despite the fact that noextensive or severe on-target toxicity against normal tis-sues has been observed, a great deal of effort has beenmade to avoid this problem. A promising strategy forthis involves the achievement of accurate tumor recogni-tion by combinatorial antigen-sensing circuits, whilebispecific antibodies have proven more specific and po-tent [108]. Another potential approach is to physicallyseparate the CD3ζ module from the costimulation mod-ule by using two distinct CARs specific for differentantigens [109–111]. This structural design allows forcomparable anticancer activity and persistence with thesecond-generation CAR-T cells only encounter both an-tigens. Another strategy is to engineer T cells with a syn-thetic Notch receptor that contains the core regulatorydomain derived from the signaling receptor Notch [112].An extracellular antigen recognition domain and a syn-thetic intracellular transcriptional domain were designedto replace the native Notch domain. Upon binding tothe first antigen, the synthetic Notch receptor is cleavedand releases the intracellular transcriptional domain toactivate the expression of the CAR, which recognizes thesecond antigen.We have noted that the immunogenicity of murine-de-

rived antibodies would limit their therapeutic effects inhumans. Similarly, the use of a CAR of murine originalso limited the persistence of CAR-T cells in recipients.The development of a CAR with a human-derived scFvis needed to address this issue. A fully human MSLN-targeting CAR (P4) was constructed and shown to beenhanced in terms of cytokine secretion and cytotoxicityin vitro and anti-tumor activity in vivo [113]. P4 CAR-Tcells were shown to be able to lyse MSLN-positivetumor cells in vitro and in vivo, even in the presence ofsoluble MSLN protein.


Table


tedtherapiesbasedon

CAR-Ttherapy

NCTNum

ber

Title

Status

Interven

tions

Phases

Enrollm

ent

Start

Date

Locatio

ns

NCT03814447

TheFourth

Gen

erationCART-cellThe

rapy

for

Refractory-Relapsed

OvarianCancer

Not

yetrecruitin

gDrug:

anti-

MESOCAR-Tcells;D

rug:

Flud

arabine;

Drug:

Cycloph

osph

amide

Early

Phase

110

2019-04-

01China

NCT03747965

Stud

yof

PD-1

Gen

e-knockedOut

Mesothe

lin-directed

CAR-TCellsWith

theCon

ditio

ning

ofPC

inMesothe

linPo

sitiveMultip

leSolid

Tumors

Recruitin

gBiolog

ical:M

esothe

lin-directed

CAR-Tcells

Phase1

102018-11-

01China

NCT03608618

Intraperito

nealMCY

-M11

(Mesothe

lin-targe

tingCAR)

forTreatm

entof

AdvancedOvarianCancerand

Periton

ealM

esothe

lioma

Recruitin

gBiolog

ical:M

CY-M

11Ph

ase1

152018-08-

27United

States

NCT03615313

PD-1

Antibod

yExpressing

mesoC

AR-TCellsfor

Mesothe

linPo

sitiveAdvancedSolid

Tumor

Recruitin

gBiolog

ical:PD-1

antib

odyexpressing

mesoC

AR-T

cells

Phase1/2

502018-08-

06China

NCT03638193

Stud

yof

Autolog

ousT-cells

inPatientsWith

Metastatic

PancreaticCancer

Recruitin

gBiolog

ical:C

ART-m

esocells

Not

App

licable

102018-07-

11China

NCT03545815

Stud

yof

CRISPR-Cas9Med

iatedPD

-1andTC

RGen

e-knocked

Out

Mesothe

lin-directed

CAR-TCellsin

PatientsWith

Mesothe

linPo

sitiveMultip

leSolid

Tumors.

Recruitin

gBiolog

ical:anti-m

esothe

linCAR-Tcells

Phase1

102018-03-

01China

NCT03356808

Antigen

-spe

cific

TCellsAgainstLung

Cancer

Recruitin

gBiolog

ical:Lun

gcancer-spe

cific

Tcells

Phase1/2

202017-12-

15China

NCT03356795

Interven

tionof

CAR-TAgainstCervicalC

ancer

Recruitin

gBiolog

ical:C

ervicalcancer-specificCAR-Tcells

Phase1/2

202017-11-

15China

NCT03497819

Autolog

ousCARTmeso/19

AgainstPancreaticCancer

Active,no

trecruitin

gBiolog

ical:C

ARTmesoCART19

Early

Phase

110

2017-10-

01China

NCT03323944

CARTCellImmun

othe

rapy

forPancreaticCancer

Recruitin

gBiolog

ical:h

uCART-m

esocells

Phase1

182017-09-

15United

States

NCT03198052

HER2/Mesothe

lin/Lew

is-Y/PSC

A/M

UC1/PD

-L1/CD80/86-CAR-T

CellsIm

mun

othe

rapy

AgainstCancers

Recruitin

gBiolog

ical:C

AR-Tcells

targetingHER2,Mesothe

lin,

PSCA,M

UC1

,Lew

is-Y,orCD80/86

Phase1

302017-07-

01China

NCT03267173

Evaluate

theSafety

andEfficacyof

CAR-Tin

theTreatm

ent

ofPancreaticCancer.

Recruitin

gDrug:

Chimericantig

enreceptor

Tcell

Early

Phase

110

2017-06-

15China

NCT03182803

CTLA-4

andPD

-1Antibod

iesExpressing

Mesothe

lin-CAR-T

CellsforMesothe

linPo

sitiveAdvancedSolid

Tumor

Recruitin

gBiolog

ical:C

TLA-4/PD-1

antib

odiesexpressing

mesoC

AR-T

Phase1/2

402017-06-

07China

NCT03054298

CARTCellsin

Mesothe

linExpressing

Cancers

Recruitin

gBiolog

ical:h

uCART-m

esocells

Phase1

302017-03-

01United

States

NCT03030001

PD-1

Antibod

yExpressing

CARTCellsforMesothe

linPo

sitiveAdvancedMalignancies

Unkno

wnstatus

Biolog

ical:PD-1

antib

odyexpressing

mesothe

linspecificCAR-Tcells

Phase1/2

402017-02-

15China

NCT02930993

Anti-m

esothe

linCARTCellsforPatientsWith

Recurren

tor

Metastatic

Malignant

Tumors

Recruitin

gBiolog

ical:anti-m

esothe

linCARTcells

Phase1

202016-08-

01China

NCT02959151

AStud

yof

ChimericAntigen

Receptor

TCellsCom

bine

dWith

Interven

tionalThe

rapy

inAdvancedLiverMalignancy

Unkno

wnstatus

Drug:

CAR-Tcell

Phase1/2

202016-07-

01China

NCT02792114

T-CellThe

rapy

forAdvancedBreastCancer

Recruitin

gDrug:

Cycloph

osph

amide;Biolog

ical:

Mesothe

lin-targe

tedTcells;D

rug:

AP1903

Phase1

362016-06-

01United

States


Table


tedtherapiesbasedon

CAR-Ttherapy(Con

tinued)

NCTNum

ber

Title

Status

Interven

tions

Phases

Enrollm

ent

Start

Date

Locatio

ns

NCT02706782

AStud

yof

Mesothe

linRedirected

Autolog

ousTCells

forAdvancedPancreaticCarcino

ma

Unkno

wnstatus

Drug:

TAI-m

eso-CART

Phase1

302016-03-

01China

NCT02580747

Treatm

entof

Relapsed

and/or

Che

mothe

rapy

Refractory

AdvancedMalignanciesby

CART-m

eso

Unkno

wnstatus

Biolog

ical:anti-m

eso-CARvector

transduced

Tcells

Phase1

202015-10-

01China

NCT02414269

Malignant

PleuralD

isease

TreatedWith

Autolog

ousT

CellsGen

eticallyEngine

ered

toTarget

theCancer-Cell

Surface

Antigen

Mesothe

lin

Recruitin

gGen

etic:iCasp9

M28zTcellinfusion

s;Drug:

cyclop

hosphamide

Phase1

482015-05-

01United

States

NCT02465983

PilotStud

yof

Autolog

ousT-cells

inPatientsWith

Metastatic

PancreaticCancer

Com

pleted

Biolog

ical:C

ART-m

eso-19

Tcells;D

rug:

Cycloph

osph

amide

Phase1

42015-05-

01United

States

NCT02388828

CART-m

esoLong

-term

Follow-up

Active,no

trecruitin

gBiolog

ical:len

tiviral-b

ased

CART

meso

therapy

102015-03-

01United

States

NCT02159716

CART-m

esoin

Mesothe

linExpressing

Cancers

Com

pleted

Biolog

ical:C

ART-m

eso

Phase1

192014-06-

01United

States

NCT01897415

Autolog

ousRedirected

RNAMesoCARTCellsfor

PancreaticCancer

Com

pleted

Biolog

ical:A

utolog

ousTcells

transfected

with

chim

ericanti-mesothe

linim

mun

orecep

tor

SS1

Phase1

162013-07-

01United

States

NCT01583686

CARTCellR

ecep

torIm

mun

othe

rapy

Targeting

Mesothe

linforPatientsWith

Metastatic

Cancer

Term

inated

Drug:

Flud

arabine;Biolog

ical:A

nti-m

esothe

linCARtransduced

PBL;Drug:

Cycolph

osph

amide;

Drug:

Aldesleukin

Phase1/2

152012-05-

04United

States

NCT01355965

Autolog

ousRedirected

RNAMeso-CIRTCells

Com

pleted

Biolog

ical:A

utolog

ousTcells

Phase1

182011-03-

01United

States


Clinical trials of MSLN-targeting CAR-T cellsThe majority of newly registered clinical trials targetingMSLN in the past 3 years are related to CAR-T therapy.CAR-T therapy has been a potent strategy for treatingMSLN-expressing tumors [86, 114]. CAR design hasbeen greatly optimized to enhance its performance [85].The safety, effects and the maximum tolerated dose ofMSLN-targeting CAR-T cell therapy are currently beingevaluated in multiple phase 1/2 clinical trials (Table 2).In a preclinical study, MSLN-targeting CAR-T cells

generated by the transfection of mRNA showed robustantitumor activity and the transient expression of theCAR. mRNA-based CAR-T cells (SS1–4-1BB CAR) wereproven to be well tolerated after multiple intravenous orintratumoral infusions (NCT01355965) [115, 116]. Aconfirmed partial response was observed in patients withMPM or PDA. The serum levels of inflammatory cyto-kines, including MIP-1β, granulocyte colony-stimulatingfactor (G-CSF), IL-6, and IL-17, were transiently elevatedafter each infusion of CAR-T cells [115]. CAR-T cellswere detected in tumors with reduced CAR transcriptsseveral days after administration. Notably, MSLN-target-ing CAR-T cells were able to lyse primary tumor cellsand elicit a systemic antitumor immune response by in-ducing epitope spreading [116].In another recent phase 1 clinical trial, 6 patients with

chemotherapy-refractory metastatic PDAC were intra-venously administered autologous MSLN-targetingCAR-T cells 3 times weekly for 3 weeks [117]. Two pa-tients had stable disease with PFS of 3.8 and 5.4 months.A decrease in MSLN expression by 69.2% in one patientwas confirmed by biopsy. None of the 6 patients experi-enced cytokine release syndrome or neurological symp-toms. Noteworthily, no evident on-target/off-tumortoxicity against normal tissues was observed in these pa-tients [116, 117]. However, in addition to the short lifespan of the CAR, another issue that might limit its po-tency is the production of human anti-CAR antibodies[115–117]. An anaphylactic response reported in one pa-tient was attributed to the high production of IgE anti-bodies specific to the CAR [115]. This suggests that afully human anti-MSLN scFv is urgently needed for clin-ical use. Interestingly, a clinical trial that aims to impedethe production of antibodies via the depletion of B cellsby CD19-targeting CAR-T cells has been initiated(NCT03497819). This clinical trial is active but is notrecruiting yet.Regional delivery was proven to enhance T cell prolif-

eration, persistence and function in mice. Because ofthis, regional delivery was applied to the clinical treat-ment of patients. CAR-T cells were administered intra-pleurally, intratumorally, or by vascular interventionalmediated injection (NCT02414269, NCT02706782,NCT02959151, NCT03267173, and NCT03198052). We

still await the publication of the clinical outcomes to de-termine the importance of regional delivery in the clinic.CAR-T therapy is always accompanied by cytokine re-

lease syndrome (CRS) and neurotoxicity due to the ex-cessive immune activation of CAR-T or non-CAR-Tcells, and the severity of this is associated with diseaseburden, the CAR-T cell dose, high-intensity lymphode-pletion and preexisting endothelial activation [118]. Todecrease the CAR-T-induced side effects, debulkingchemotherapy is recommended to reduce tumor burdenand the subsequent CAR-T dose, and tocilizumab couldbe used to prevent severe CRS in the clinic [118]. To en-hance the safety of CAR-T therapy and controllablyeliminate CAR-T cells when SAEs occur or tumors areeliminated, inducible suicide genes, including iCaspase-9, HSV-TK or EGFRΔ, could co-transduced with theCAR genes [25]. Exposure to a synthetic dimerizing drugwould induce the dimerization of iCaspase-9 and lead tocell apoptosis. This inducible T-cell safety switch involv-ing iCaspase-9 has been proven to eliminate over 90% ofmodified T cells within 30 min [119]. A MSLN-targetingCAR-T therapy trial involving the use of iCaspase-9 iscurrently recruiting (NCT03747965).

ConclusionsThe expression pattern of MSLN provides an excitingopportunity for its use in targeted therapy in varioustypes of malignant tumors, including pancreatic cancer,ovarian cancer, lung cancer, TNBC and gastric cancer.To date, antibody-based drugs have been effective ininhibiting cancer progression and show acceptable “ontarget, off tumor” toxicity, while vaccines have showedmoderate effects. The great improvements in CAR-T de-sign allows them to be a promising therapeutic strategyto treat MSLN-expressing tumors. The immunogenicityof drugs and CAR-T cells, the low level of T cell infiltra-tion into tumors and the high level of immunosuppres-sion in the tumor microenvironment are obstacles thatneed to be overcome. The combined use with check-point inhibitors as well as additional strategies to reducedrug resistance and optimize delivery regimens mightshow promise in the future.

AbbreviationsADCC: Antibody-dependent cell-mediated cytotoxicity; ADCP: Antibody-dependent cell-mediated phagocytosis; cfu: Colony forming units;CRS: Cytokine release syndrome; DHAEs: Drug hypersensitivity adverseevents; DLTs: Dose-limiting toxicities; G-CSF: Granulocyte colony-stimulatingfactor; GPI: Glycosylphosphatidylinositol; Lm: Listeria monocytogenes;MMAE: Monomethyl auristatin E; MPF: Megakaryocyte potentiating factor;MPM: Malignant pleural mesothelioma; MSLN: Mesothelin; MTD: Maximumtolerated dose; NSCLC: Non-small cell lung cancer; PD: Progressive disease;PDA: Pancreatic ductal carcinoma; PE: Pseudomonas exotoxin;PK: Pharmacokinetics; PLD: Pegylated liposomal doxorubicin; PR: Partialresponses; RIT: Recombination immunotoxin; scFv: Single chain variablefragment; SD: Stable disease; SMRP: Soluble MSLN-related protein;TNBC: Triple negative breast cancer


AcknowledgementsNot applicable.

Authors’ contributionsWriting, review, and/or revision of the manuscript: JL, PL. Both authors readand approved the final manuscript.

FundingThis study is supported by Guangzhou science and technology plan project,NO. 201907010042, 201904010473; The National Major Scientific andTechnological Special Project for “Significant New Drugs Development”, No.SQ2018ZX090201; The Strategic Priority Research Program of the ChineseAcademy of Sciences, Grant No. XDB19030205, No. XDA12050305;Guangdong Provincial Applied Science and Technology Research &Development Program, No. 2016B020237006; Guangdong Special SupportProgram, NO. 2017TX04R102; Frontier and key technology innovation specialgrant from the Department of Science and Technology of Guangdongprovince, No. 2015B020227003; Natural Science Fund of GuangdongProvince: Distinguished Young Scholars (Grant No.: 2014A030306028),Doctoral Foundation, No.: 2017A030310381; National Natural ScienceFoundation of China (NSFC), No. 81773301; 81700156; 81870121; 81873847;Frontier Research Program of Guangzhou Regenerative Medicine and HealthGuangdong Laboratory, No. 2018GZR110105003; Science and TechnologyPlanning Project of Guangdong Province, China (2017B030314056);Guangzhou Medical University High-level University Construction ResearchStartup Fund, NO. B195002004013; Research Program of Hefei Institute ofStem Cell and Regenerative Medicine, No. 2019YF001.

Availability of data and materialsNot applicable.

Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Key Laboratory of Regenerative Biology, South China Institute for Stem CellBiology and Regenerative Medicine, Guangzhou Institutes of Biomedicineand Health, Chinese Academy of Sciences, Guangzhou, China. 2GuangdongProvincial Key Laboratory of Stem Cell and Regenerative Medicine, SouthChina Institute for Stem Cell Biology and Regenerative Medicine, GuangzhouInstitutes of Biomedicine and Health, Chinese Academy of Sciences,Guangzhou, China. 3University of Chinese Academy of Sciences, ShijingshanDistrict, Beijing, China.

Received: 12 June 2019 Accepted: 31 July 2019

References1. Yamaguchi N, Hattori K, Oh-eda M, Kojima T, Imai N, Ochi N. A novel

cytokine exhibiting megakaryocyte potentiating activity from a humanpancreatic tumor cell line HPC-Y5. J Biol Chem. 1994;269(2):805–8.

2. Chang K, Pastan I, Willingham MC. Isolation and characterization of amonoclonal antibody, K1, reactive with ovarian cancers and normalmesothelium. Int J Cancer. 1992;50(3):373–81.

3. Ordonez NG. Application of mesothelin immunostaining in tumor diagnosis.Am J Surg Pathol. 2003;27(11):1418–28.

4. Bera TK, Pastan I. Mesothelin is not required for normal mousedevelopment or reproduction. Mol Cell Biol. 2000;20(8):2902–6.

5. Servais EL, Colovos C, Rodriguez L, Bograd AJ, Nitadori J, Sima C, Rusch VW,Sadelain M, Adusumilli PS. Mesothelin overexpression promotesmesothelioma cell invasion and MMP-9 secretion in an orthotopic mousemodel and in epithelioid pleural mesothelioma patients. Clin Cancer Res.2012;18(9):2478–89.

6. Rump A, Morikawa Y, Tanaka M, Minami S, Umesaki N, Takeuchi M, MiyajimaA. Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediatescell adhesion. J Biol Chem. 2004;279(10):9190–8.

7. Gubbels JA, Belisle J, Onda M, Rancourt C, Migneault M, Ho M, Bera TK,Connor J, Sathyanarayana BK, Lee B, Pastan I, Patankar MS. Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction thatfacilitates peritoneal metastasis of ovarian tumors. Mol Cancer. 2006;5(1):50.

8. Coelho R, Marcos-Silva L, Ricardo S, Ponte F, Costa A, Lopes JM, DavidL. Peritoneal dissemination of ovarian cancer: role of MUC16-mesothelininteraction and implications for treatment. Expert Rev Anticancer Ther.2018;18(2):177–86.

9. Chen SH, Hung WC, Wang P, Paul C, Konstantopoulos K. Mesothelin bindingto CA125/MUC16 promotes pancreatic cancer cell motility and invasion viaMMP-7 activation. Sci Rep. 2013;3:1870.

10. Bharadwaj U, Marin-Muller C, Li M, Chen C, Yao Q. Mesothelin conferspancreatic cancer cell resistance to TNF-alpha-induced apoptosisthrough Akt/PI3K/NF-kappaB activation and IL-6/Mcl-1 overexpression.Mol Cancer. 2011;10:106.

11. Kachala SS, Bograd AJ, Villena-Vargas J, Suzuki K, Servais EL, Kadota K, ChouJ, Sima CS, Vertes E, Rusch VW, Travis WD, Sadelain M, Adusumilli PS.Mesothelin overexpression is a marker of tumor aggressiveness and isassociated with reduced recurrence-free and overall survival in early-stagelung adenocarcinoma. Clin Cancer Res. 2014;20(4):1020–8.

12. Tozbikian G, Brogi E, Kadota K, Catalano J, Akram M, Patil S, Ho AY,Reis-Filho JS, Weigelt B, Norton L, Adusumilli PS, Wen HY. Mesothelinexpression in triple negative breast carcinomas correlates significantlywith basal-like phenotype, distant metastases and decreased survival.PLoS One. 2014;9(12):e114900.

13. Sathyanarayana BK, Hahn Y, Patankar MS, Pastan I, Lee B. Mesothelin,Stereocilin, and Otoancorin are predicted to have superhelical structureswith ARM-type repeats. BMC Struct Biol. 2009;9:1.

14. Ma J, Tang WK, Esser L, Pastan I, Xia D. Characterization of crystals of anantibody-recognition fragment of the cancer differentiation antigenmesothelin in complex with the therapeutic antibody MORAb-009. ActaCrystallogr Sect F Struct Biol Cryst Commun. 2012;68(Pt 8):950–3.

15. Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigenpresent on mesothelium, mesotheliomas, and ovarian cancers. Proc NatlAcad Sci U S A. 1996;93(1):136–40.

16. Chang K, Pai LH, Pass H, Pogrebniak HW, Tsao MS, Pastan I, Willingham MC.Monoclonal antibody K1 reacts with epithelial mesothelioma but not withlung adenocarcinoma. Am J Surg Pathol. 1992;16(3):259–68.

17. Ordonez NG. Value of mesothelin immunostaining in the diagnosis ofmesothelioma. Mod Pathol. 2003;16(3):192–7.

18. Miettinen M, Sarlomo-Rikala M. Expression of calretinin,thrombomodulin, keratin 5, and mesothelin in lung carcinomas ofdifferent types: an immunohistochemical analysis of 596 tumors incomparison with epithelioid mesotheliomas of the pleura. Am J SurgPathol. 2003;27(2):150–8.

19. Argani P, Iacobuzio-Donahue C, Ryu B, Rosty C, Goggins M, Wilentz RE,Murugesan SR, Leach SD, Jaffee E, Yeo CJ, Cameron JL, Kern SE, Hruban RH.Mesothelin is overexpressed in the vast majority of ductal adenocarcinomasof the pancreas: identification of a new pancreatic cancer marker by serialanalysis of gene expression (SAGE). Clin Cancer Res. 2001;7(12):3862–8.

20. Hassan R, Laszik ZG, Lerner M, Raffeld M, Postier R, Brackett D. Mesothelin isoverexpressed in pancreaticobiliary adenocarcinomas but not in normalpancreas and chronic pancreatitis. Am J Clin Pathol. 2005;124(6):838–45.

21. O'Hara MH, Stashwick C, Plesa G, Tanyi JL. Overcoming barriers of car T-celltherapy in patients with mesothelin-expressing cancers. Immunotherapy.2017;9(9):767–80.

22. Chang K, Pastan I, Willingham MC. Frequent expression of the tumorantigen CAK1 in squamous-cell carcinomas. Int J Cancer. 1992;51(4):548–54.

23. Tchou J, Wang LC, Selven B, Zhang H, Conejo-Garcia J, Borghaei H, Kalos M,Vondeheide RH, Albelda SM, June CH, Zhang PJ. Mesothelin, a novelimmunotherapy target for triple negative breast cancer. Breast Cancer ResTreat. 2012;133(2):799–804.

24. Rizk NP, Servais EL, Tang LH, Sima CS, Gerdes H, Fleisher M, Rusch VW,Adusumilli PS. Tissue and serum mesothelin are potential markers ofneoplastic progression in Barrett's associated esophageal adenocarcinoma.Cancer Epidemiol Biomark Prev. 2012;21(3):482–6.

25. Morello A, Sadelain M, Adusumilli PS. Mesothelin-targeted CARs: driving Tcells to solid tumors. Cancer Discov. 2016;6(2):133–46.


26. Pastan I, Hassan R. Discovery of mesothelin and exploiting it as a target forimmunotherapy. Cancer Res. 2014;74(11):2907–12.

27. Han SH, Joo M, Kim H, Chang S. Mesothelin expression in gastricadenocarcinoma and its relation to clinical outcomes. J Pathol Transl Med.2017;51(2):122–8.

28. Lv J, Zhao R, Wu D, Zheng D, Wu Z, Shi J, Wei X, Wu Q, Long Y, Lin S, WangS, Wang Z, Li Y, Chen Y, He Q, Chen S, Yao H, Liu Z, Tang Z, Yao Y, Pei D,Liu P, Zhang X, Zhang Z, Cui S, Chen R, Li P. Mesothelin is a target ofchimeric antigen receptor T cells for treating gastric cancer. J HematolOncol. 2019;12(1):18.

29. Cheng WF, Huang CY, Chang MC, Hu YH, Chiang YC, Chen YL, Hsieh CY,Chen CA. High mesothelin correlates with chemoresistance and poorsurvival in epithelial ovarian carcinoma. Br J Cancer. 2009;100(7):1144–53.

30. Kawamata F, Kamachi H, Einama T, Homma S, Tahara M, Miyazaki M, TanakaS, Kamiyama T, Nishihara H, Taketomi A, Todo S. Intracellular localization ofmesothelin predicts patient prognosis of extrahepatic bile duct cancer. Int JOncol. 2012;41(6):2109–18.

31. Nomura R, Fujii H, Abe M, Sugo H, Ishizaki Y, Kawasaki S, Hino O. Mesothelinexpression is a prognostic factor in cholangiocellular carcinoma. Int Surg.2013;98(2):164–9.

32. Thomas A, Chen Y, Steinberg SM, Luo J, Pack S, Raffeld M, Abdullaev Z,Alewine C, Rajan A, Giaccone G, Pastan I, Miettinen M, Hassan R. Highmesothelin expression in advanced lung adenocarcinoma is associated withKRAS mutations and a poor prognosis. Oncotarget. 2015;6(13):11694–703.

33. Li YR, Xian RR, Ziober A, Conejo-Garcia J, Perales-Puchalt A, June CH, ZhangPJ, Tchou J. Mesothelin expression is associated with poor outcomes inbreast cancer. Breast Cancer Res Treat. 2014;147(3):675–84.

34. Winter JM, Tang LH, Klimstra DS, Brennan MF, Brody JR, Rocha FG, Jia X, QinLX, D'Angelica MI, DeMatteo RP, Fong Y, Jarnagin WR, O'Reilly EM, Allen PJ.A novel survival-based tissue microarray of pancreatic cancer validatesMUC1 and mesothelin as biomarkers. PLoS One. 2012;7(7):e40157.

35. Shimizu A, Hirono S, Tani M, Kawai M, Okada K, Miyazawa M, Kitahata Y,Nakamura Y, Noda T, Yokoyama S, Yamaue H. Coexpression of MUC16 andmesothelin is related to the invasion process in pancreatic ductaladenocarcinoma. Cancer Sci. 2012;103(4):739–46.

36. Hassan R, Thomas A, Alewine C, Le DT, Jaffee EM, Pastan I. Mesothelinimmunotherapy for Cancer: ready for prime time? J Clin Oncol. 2016;34(34):4171–9.

37. Cristaudo A, Bonotti A, Guglielmi G, Fallahi P, Foddis R. Serum mesothelinand other biomarkers: what have we learned in the last decade? J ThoracDis. 2018;10(Suppl 2):S353–s359.

38. Sapede C, Gauvrit A, Barbieux I, Padieu M, Cellerin L, Sagan C, ScherpereelA, Dabouis G, Gregoire M. Aberrant splicing and protease involvement inmesothelin release from epithelioid mesothelioma cells. Cancer Sci. 2008;99(3):590–4.

39. Robinson BW, Creaney J, Lake R, Nowak A, Musk AW, de Klerk N, Winzell P,Hellstrom KE, Hellstrom I. Mesothelin-family proteins and diagnosis ofmesothelioma. Lancet. 2003;362(9396):1612–6.

40. Bast RC Jr. Status of tumor markers in ovarian cancer screening. J ClinOncol. 2003;21(10 Suppl):200s–5s.

41. Hassan R, Wu C, Brechbiel MW, Margulies I, Kreitman RJ, Pastan I.111Indium-labeled monoclonal antibody K1: biodistribution study in nudemice bearing a human carcinoma xenograft expressing mesothelin. Int JCancer. 1999;80(4):559–63.

42. Hassan R, Viner JL, Wang QC, Margulies I, Kreitman RJ, Pastan I. Anti-tumoractivity of K1-LysPE38QQR, an immunotoxin targeting mesothelin, a cell-surface antigen overexpressed in ovarian cancer and malignantmesothelioma. J Immunother. 2000;23(4):473–9.

43. Chowdhury PS, Viner JL, Beers R, Pastan I. Isolation of a high-affinity stablesingle-chain Fv specific for mesothelin from DNA-immunized mice byphage display and construction of a recombinant immunotoxin with anti-tumor activity. Proc Natl Acad Sci U S A. 1998;95(2):669–74.

44. Chowdhury PS, Pastan I. Improving antibody affinity by mimicking somatichypermutation in vitro. Nat Biotechnol. 1999;17(6):568–72.

45. Hilliard TS. The Impact of Mesothelin in the Ovarian Cancer TumorMicroenvironment. Cancers (Basel). 2018;10(9):277.

46. Hassan R, Sharon E, Thomas A, Zhang J, Ling A, Miettinen M, Kreitman RJ,Steinberg SM, Hollevoet K, Pastan I. Phase 1 study of the antimesothelinimmunotoxin SS1P in combination with pemetrexed and cisplatin for front-line therapy of pleural mesothelioma and correlation of tumor responsewith serum mesothelin, megakaryocyte potentiating factor, and cancerantigen 125. Cancer. 2014;120(21):3311–9.

47. Hassan R, Miller AC, Sharon E, Thomas A, Reynolds JC, Ling A, Kreitman RJ,Miettinen MM, Steinberg SM, Fowler DH, Pastan I. Major cancer regressionsin mesothelioma after treatment with an anti-mesothelin immunotoxin andimmune suppression. Sci Transl Med. 2013;5(208):208ra147.

48. Hassan R, Bullock S, Premkumar A, Kreitman RJ, Kindler H, Willingham MC,Pastan I. Phase I study of SS1P, a recombinant anti-mesothelinimmunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers. Clin Cancer Res.2007;13(17):5144–9.

49. Kreitman RJ, Hassan R, Fitzgerald DJ, Pastan I. Phase I trial of continuousinfusion anti-mesothelin recombinant immunotoxin SS1P. Clin Cancer Res.2009;15(16):5274–9.

50. Gupta A, Hussein Z, Hassan R, Wustner J, Maltzman JD, Wallin BA.Population pharmacokinetics and exposure-response relationship ofamatuximab, an anti-mesothelin monoclonal antibody, in patients withmalignant pleural mesothelioma and its application in dose selection.Cancer Chemother Pharmacol. 2016;77(4):733–43.

51. Fujisaka Y, Kurata T, Tanaka K, Kudo T, Okamoto K, Tsurutani J, Kaneda H,Okamoto I, Namiki M, Kitamura C, Nakagawa K. Phase I study ofamatuximab, a novel monoclonal antibody to mesothelin, in Japanesepatients with advanced solid tumors. Investig New Drugs. 2015;33(2):380–8.

52. Hassan R, Cohen SJ, Phillips M, Pastan I, Sharon E, Kelly RJ, Schweizer C, WeilS, Laheru D. Phase I clinical trial of the chimeric anti-mesothelin monoclonalantibody MORAb-009 in patients with mesothelin-expressing cancers. ClinCancer Res. 2010;16(24):6132–8.

53. Hassan R, Schweizer C, Lu KF, Schuler B, Remaley AT, Weil SC, Pastan I.Inhibition of mesothelin-CA-125 interaction in patients with mesotheliomaby the anti-mesothelin monoclonal antibody MORAb-009: implications forcancer therapy. Lung Cancer. 2010;68(3):455–9.

54. Hassan R, Kindler HL, Jahan T, Bazhenova L, Reck M, Thomas A, Pastan I,Parno J, O'Shannessy DJ, Fatato P, Maltzman JD, Wallin BA. Phase II clinicaltrial of amatuximab, a chimeric antimesothelin antibody with pemetrexedand cisplatin in advanced unresectable pleural mesothelioma. Clin CancerRes. 2014;20(23):5927–36.

55. Quanz M, Hagemann UB, Zitzmann-Kolbe S, Stelte-Ludwig B, Golfier S, ElbiC, Mumberg D, Ziegelbauer K, Schatz CA. Anetumab ravtansine inhibitstumor growth and shows additive effect in combination with targetedagents and chemotherapy in mesothelin-expressing human ovarian cancermodels. Oncotarget. 2018;9(75):34103–21.

56. Golfier S, Kopitz C, Kahnert A, Heisler I, Schatz CA, Stelte-Ludwig B, Mayer-Bartschmid A, Unterschemmann K, Bruder S, Linden L, Harrenga A, Hauff P,Scholle FD, Muller-Tiemann B, Kreft B, Ziegelbauer K. Anetumab ravtansine:a novel mesothelin-targeting antibody-drug conjugate cures tumors withheterogeneous target expression favored by bystander effect. Mol CancerTher. 2014;13(6):1537–48.

57. Baldo P, Cecco S. Amatuximab and novel agents targeting mesothelin forsolid tumors. Onco Targets Ther. 2017;10:5337–53.

58. Le DT, Wang-Gillam A, Picozzi V, Greten TF, Crocenzi T, Springett G, MorseM, Zeh H, Cohen D, Fine RL, Onners B, Uram JN, Laheru DA, Lutz ER, Solt S,Murphy AL, Skoble J, Lemmens E, Grous J, Dubensky T Jr, Brockstedt DG,Jaffee EM. Safety and survival with GVAX pancreas prime and Listeriamonocytogenes-expressing mesothelin (CRS-207) boost vaccines formetastatic pancreatic cancer. J Clin Oncol. 2015;33(12):1325–33.

59. Le DT, Brockstedt DG, Nir-Paz R, Hampl J, Mathur S, Nemunaitis J, Sterman DH,Hassan R, Lutz E, Moyer B, Giedlin M, Louis JL, Sugar EA, Pons A, Cox AL, LevineJ, Murphy AL, Illei P, Dubensky TW Jr, Eiden JE, Jaffee EM, Laheru DA. A live-attenuated Listeria vaccine (ANZ-100) and a live-attenuated Listeria vaccineexpressing mesothelin (CRS-207) for advanced cancers: phase I studies ofsafety and immune induction. Clin Cancer Res. 2012;18(3):858–68.

60. Lindenberg L, Thomas A, Adler S, Mena E, Kurdziel K, Maltzman J, Wallin B,Hoffman K, Pastan I, Paik CH, Choyke P, Hassan R. Safety and biodistributionof 111In-amatuximab in patients with mesothelin expressing cancers usingsingle photon emission computed tomography-computed tomography(SPECT-CT) imaging. Oncotarget. 2015;6(6):4496–504.

61. Scales SJ, Gupta N, Pacheco G, Firestein R, French DM, Koeppen H, RangellL, Barry-Hamilton V, Luis E, Chuh J, Zhang Y, Ingle GS, Fourie-O'Donohue A,Kozak KR, Ross S, Dennis MS, Spencer SD. An antimesothelin-monomethylauristatin e conjugate with potent antitumor activity in ovarian, pancreatic,and mesothelioma models. Mol Cancer Ther. 2014;13(11):2630–40.

62. Weekes CD, Lamberts LE, Borad MJ, Voortman J, McWilliams RR, DiamondJR, de Vries EG, Verheul HM, Lieu CH, Kim GP, Wang Y, Scales SJ, Samineni


D, Brunstein F, Choi Y, Maslyar DJ, Colon-Otero G. Phase I study ofDMOT4039A, an antibody-drug conjugate targeting Mesothelin, in patientswith Unresectable pancreatic or platinum-resistant ovarian Cancer. MolCancer Ther. 2016;15(3):439–47.

63. Hollevoet K, Mason-Osann E, Liu XF, Imhof-Jung S, Niederfellner G, Pastan I. Invitro and in vivo activity of the low-immunogenic antimesothelin immunotoxinRG7787 in pancreatic cancer. Mol Cancer Ther. 2014;13(8):2040–9.

64. El-Behaedi S, Landsman R, Rudloff M, Kolyvas E, Albalawy R, Zhang X, Bera T,Collins K, Kozlov S, Alewine C. Protein Synthesis Inhibition Activity ofMesothelin Targeting Immunotoxin LMB-100 Decreases Concentrations ofOncogenic Signaling Molecules and Secreted Growth Factors. Toxins (Basel).2018;10(11):447.

65. Ali-Rahmani F, FitzGerald DJ, Martin S, Patel P, Prunotto M, OrmanogluP, Thomas C, Pastan I. Anticancer effects of Mesothelin-targetedimmunotoxin therapy are regulated by tyrosine kinase DDR1. CancerRes. 2016;76(6):1560–8.

66. Alewine C, Xiang L, Yamori T, Niederfellner G, Bosslet K, Pastan I. Efficacy ofRG7787, a next-generation mesothelin-targeted immunotoxin, against triple-negative breast and gastric cancers. Mol Cancer Ther. 2014;13(11):2653–61.

67. Liu XF, Xiang L, Zhou Q, Carralot JP, Prunotto M, Niederfellner G, Pastan I.Actinomycin D enhances killing of cancer cells by immunotoxin RG7787through activation of the extrinsic pathway of apoptosis. Proc Natl Acad SciU S A. 2016;113(38):10666–71.

68. Zhang J, Khanna S, Jiang Q, Alewine C, Miettinen M, Pastan I, Hassan R.Efficacy of anti-mesothelin immunotoxin RG7787 plus nab-paclitaxel againstmesothelioma patient-derived xenografts and Mesothelin as a biomarker oftumor response. Clin Cancer Res. 2017;23(6):1564–74.

69. Kolyvas E, Rudloff M, Poruchynsky M, Landsman R, Hollevoet K, Venzon D,Alewine C. Mesothelin-targeted immunotoxin RG7787 has synergistic anti-tumor activity when combined with taxanes. Oncotarget. 2017;8(6):9189–99.

70. Liu XF, Zhou Q, Hassan R, Pastan I. Panbinostat decreases cFLIP andenhances killing of cancer cells by immunotoxin LMB-100 by stimulatingthe extrinsic apoptotic pathway. Oncotarget. 2017;8(50):87307–16.

71. Wickstroem K, Hagemann UB, Cruciani V, Wengner AM, Kristian A, EllingsenC, Siemeister G, Bjerke R, Karlsson J, Ryan OB, Linden L, Mumberg D,Ziegelbauer K, Cuthbertson AS. Synergistic effect of a Mesothelin targetedThorium-227 conjugate in combination with DNA damage responseinhibitors in ovarian Cancer xenograft models. J Nucl Med. 2019. https://www.ncbi.nlm.nih.gov/pubmed/30850485. http://jnm.snmjournals.org/citmgr?gca=jnumed%3Bjnumed.118.223701v1.

72. Richard Austin WA, Patrick A. Baeuerle, Adrie Jones, S

mesothelin as a biomarker for targeted therapy...expression is generally limited to normal...

Documents